Fig 2: Comparison of the expression and activity of Dusp5 in WBCs isolated from Dusp5 ZFN KO versus FHH.1BN rats. Panel A: The expression of Dusp5 protein in WBCs is nearly absent in Dusp5 KO versus FHH.1BN rats. The levels of phosphorylated-ERK1/2 protein are significantly increased in Dusp5 KO compared to FHH.1BN rats, but there is no change in the expression of total ERK or beta-Actin. Panel B presents the results of the expression of Dusp5, p-ERK1/2, total ERK1/2 and β-Actin protein from duplicate aliquots of a single pooled microvessel sample isolated from 8–10 Dusp5 KO and FHH.1BN rats. The upper panel presents a representative image of the gels and the lower panel presents the quantitation of the images above. Mean values ± SE from 3 rats per strain are presented in Panel A. * indicates a significant difference from the corresponding value in Dusp5 KO rats.

Fig 3: Schematic describing the generation of Dusp5 ZFN KO rats in the FHH.1BN genetic background. Panel A presents the sequence of the Dusp5 ZFN. The Dusp5 specific ZFN introduced a 14 bp deletion and a 3 bp insertion resulting in a net 11 bp deletion between nucleotides 449–464 in the Dusp5 sequence in the KO animals that introduced a frame shift mutation. Panel B presents the strategy for the generation of Dusp5 ZFN KO rats in the FHH.1BN genetic background. Fertilized donor embryos from female FHH.1BN rats were collected and the Dusp5 ZFN mRNA was microinjected into pronuclei of the fertilized one-cell embryos. These embryos were transferred back to a foster mother. The heterozygous founders were brother-sister mated to generate the homozygous ZFN KO founders and the FHH.1BN wild type control rats. Panel C presents an example of PCR genotyping of the region of interest in FHH.1BN and Dusp5 KO rats. The rats were genotyped using the following primers: Dusp5-F: 5'-GCT GCA GGA GGG CGG CGG CG -3', Dusp5 R: 5'-CTT TAA GGA AGT AGA CCC G-3'. These primers amplify a 155 bp band for the wild type allele and a 144 bp band for the knockout allele. Both bands are observed in heterozygous rats.

Fig 4: Identification of the Zn-finger target site and deletion in the Dusp5 KO strain. Panel A presents a schematic model of the Dusp5 protein. The Dusp5 Zinc-finger construct targets amino acids (AA) 92–96 in the N-terminal regulatory rhodanese domain (AA5-140) resulting in the introduction of a premature stop codon at AA121 that is predicted to produce a truncated protein. The Dusp5 antibody used in these studies targets AA286-384 in the C-terminal phosphatase catalytic domain (AA178-314). Panel B presents a comparison of I-TASSER predicted structure and the folding of the Dusp5 protein in FHH (155R) and FHH.1BN (155G) rats. The upper panels show the predicted structure of the Dusp5 protein in both strains based on the complete AA sequence. The putative catalytic triad (Asp232/Ser268/Cys263) is shown in a “stick figure” form and the 3-letter AA codes are labeled in black. The rest of the protein is represented as ribbon running along the backbone. Secondary structural elements are depicted by color with helices, beta sheets and coils represented in red, cyan and white, respectively. The putative catalytic triad is magnified and shown in “stick figure” form in the lower panel and the 3-letter AA codes are labeled with in black. Only residues 174–320 of Dusp5 protein are presented in order to enhance the view of the putative catalytic triad. There are significant structural differences both in the overall folding of the Dusp5 protein that impact on the structure of the active site/catalytic triad region between the strains. This may account for the observed differences in the activity of the Dusp5 protein in FHH versus FHH.1BN rats.

Fig 5: Comparison of autoregulation of CBF in Dusp5 ZFN KO versus FHH.1BN and FHH rats.The relationships between cerebral blood flow and mean arterial pressures in 9–12 week old Dusp5 ZFN KO, FHH.1BN, FHH and Sprague Dawley rats are compared. Mean values ± SE are presented. * indicates significantly difference in the corresponding value in FHH rats versus all the other strains. # indicates a significant difference in the corresponding values in Dusp5 KO and FHH.1BN rats. Numbers in parentheses indicate numbers of animal studied per strain.